Due to various properties of fusion proteins, the ratio of HRV 3C protease: target protein, temperature, incubation time is recommended to be optimized for practical application.

The following protocol is a simple example to estimate the appropriate amount of the enzyme.

Combine 100μg fusion protein, 10μL 10×HRV 3C protease Cleavage Buffer, HRV 3C protease of different volumes and sterile water to make a 100μL total reaction volume. A control sample without HRV 3C protease should be included to detect a possible unspecific cleavage either by autolysis or by proteolytic contaminations of the fusion protein.

Component

Volume (μL)

enzyme volume (μL)

X

100μg control protein

Y

10X Cleavage buffer

10

Sterile water

100-X-Y

Incubate the reaction mixture at 4℃ for 16 hours or overnight.

Take out 20μL sample and add 20μL 2×SDS-PAGE loading buffer for each treatment and store at -20℃ until SDS-PAGE analysis. If practical, take out aliquots at different time spots to optimize the incubation time.

Determine and compare the extent of cleavage of the samples by SDS-PAGE analysis.

The protease: target protein ratio can reach 1:800 in our experiment, but we recommend excessive use of the high specificity protease(1:100~1:25) to insure enzymatic digest completely in different experiments.

If shorter incubation time is required, more amount of HRV 3C protease or higher temperature (RT) can be implemented.

When the cleavage conditions are optimized at a small scale, scale up the cleavage proportionally according to specific application requirement.

If IMAC Ni-charged resin is used after cleavage to remove the HRV 3C protease, the buffer of target protein should be exchanged into suitable buffers without EDTA or imidazole. Buffer exchange can be carried out by desalting or dialysis.

Fig. The control protein was cleaved by HRV 3C protease at 4℃ for 16 h.

Additional information

Molecular weight:

~22KDa on SDS-PAGE

SDS-PAGE:

Purity:

98% by SDS-PAGE.

Quality control:

The purity of each lot is determined by SDS-PAGE. and the activity is ensured by cleavage test with a recombinant fusion protein for each lot. The solution of HRV 3C protease is filtered through 0.22μm sterile filter before package.

Factors that influence HRV 3C activity

Depending on the buffers used and their chemical components, HRV 3C Protease cleavage efficiency may be affected. The following table shows the relative activity of HRV 3C Protease under various conditions.

Factor

Reagent

Concentration

Relative Activity (%)

Salt

NaCl

0.8M

150

0.2M

110

2.5~3M

200

ZnCl2

0.2mM

0

Na2SO4

0.8M

1570~7200

Protease inhibitor

EDTA

50mM

100

EGTA

50mM

100

Egg White cystatin

8μM

100

E-64

100μM

100

Iodoacetamide

1.0±0.1mM

50

Pepstatin

20μM

100

Aprotinin

15μM

100

Benzamidine

50mM

100

Leupeptin

0.75±0.05mM

50

PMSF

8.0±0.2mM

50

TLCK

>1.0mM

50

Denaturant

Urea

3M

0

2M

0

1M

40

Guanadine

3M

0

2M

0

1M

0

Reductant

DTT

1mM

100

Detergent

Triton X-100

0.10%

>100

1%

100

Tween 20

0.10%

>100

1%

100

Nonidet P-40

0.10%

>100

1%

100

Anion(Na salt)

F-

0.2M

250

0.4M

470

Cl-

0.2M

110

0.4M

130

0.8M

150

Br-

0.2M

90

0.4M

85

0.8M

81

I-

0.2M

81

0.4M

63

0.8M

54

CH3CO2-

0.2M

150

0.4M

181

0.8M

338

SO32-

0.2M

122

0.4M

220

0.8M

365

SO42-

0.2M

252

0.4M

680

0.8M

1570

1M

2200

Co-solvent

Acetonitrile

10%

48

DMSO

10%

74

Isopropanol

10%

74

Methanol

10%

91

Glycerol

10%

114

Etylene glycol

10%

95

PEG-3400

10%

90

Sorbitol

10%

120

Sucrose

10%

112

Elute buffer

Imidazole

20~250mM

100

Troubleshooting guide

Problem

Probable cause

Solution

Incomplete cleavage

Suboptimal HRV 3C Protease to fusion protein ratio

Confirm the amount of fusion protein in the digestion. Adjust the amount of HRV 3C Protease added to at least 10 U/mg fusion protein.

Insufficient incubation period

Increase reaction time.

HRV 3C Protease recognition site not present or has been altered during the course of cloning.

Verify presence of optimal HRV 3C Protease cleavage sequence.

HRV 3C Protease recognition site is not accessible.

Reversibly denature protein with non-ionic detergents, denaturants .

HRV 3C Protease inhibitors present

Dialyze the fusion protein against Cleavage Buffer before cleaving with HRV 3C Protease.

Use protease-deficient strain (e.g., lon or ompT), such as E. coli BL21(DE3).

Background information

The catalytic activities of 3C protease have been found to be essential for generation of mature viral proteins and functional viral enzymes and thus for viral replication in the human rhinoviruses (HRVs) which are members of the picornavirus family and are the major causative agents of common cold. HRVs contain a single strand, positive-sense RNA genome which encodes a single viral polyprotein. This polyprotein precursor is further processed by two viral proteases designated 2A and 3C protease. It is believed that the 2A protease makes the first cleavage at its own N-terminus to separate the structural capsid proteins from the nonstructural ones, while the 3C protease processes most of the remaining sites.

Structure of HRV 3C protease

Sequence comparison, structure and mutational analysis, and studies with classic protease inhibitors have all shown that HRV 3C protease contains an active site cysteine residue as the nucleophile. However, its overall structures are more like serine proteases rather than the typical cysteine proteases. For HRV 3C protease, the catalytic triad is composed of His-Glu-Cys and is positioned as seen in serine proteases. HRV 3C protease will fold into two anti-parallel six-stranded β-barrels and the site cleft is located at the junction of the two β-barrels domains.

Activity of HRV 3C protease

In infected cells, HRV14 3C is able to process the polyprotein precursor at the scissile bonds formed between Gln-Gly, Gln-Ala, and Glu-Gly. However, in vitro studies have shown that this enzyme has a robust activity to cleave at the PreScission site (Leu-Glu-Val-Leu-Phe-Gln-↓-Gly-Pro) with high specificity. And the enzyme requires neither metal nor cofactors for activity. It has been demonstrated that the enzyme exhibits highest activity around neutral pH at temperatures ranging from 22 to 37℃, even retaining robust activity at 4℃.

Recombinant HRV 3C protease

Recombinant HRV 3C protease encoded by human rhinovirus 14 is a highly purified recombinant protease with a His-tag or GST-tag produced in E.coli system which has the activity as same as the wild type. The protease is a ~20kDa single-chain protein containing approximately 182 amino acids cover the 1538-1717 region of HRV 14's Genome polyprotein (Accession #: P03303) with calculated isoelectric point 8.46.